Method for extracting proteins

ABSTRACT

The present invention relates to a method and a kit for the sequential extraction of proteins for the production of partial proteomes from a complete proteome. In particular, the method according to the invention, besides other partial proteomes, gives a partial proteome of proteins from the cell nucleus interior separated from proteins of the cytoskeleton and of the nuclear matrix.

The invention relates to a method for the extraction of cellularproteins depending on their subcellular localisation (topology). Inparticular, the method according to the invention facilitates theisolation of proteins from the cell nucleus interior as an independentfraction by separating the complete proteome into partial proteomes andthus makes this fraction available for further analyses for the firsttime in adequate purity and quality.

With the sequencing of the human genome, science has gained access tothe individual genetic code of each human being. This providesinformation on his/her descent and origin. However, this information isinadequate for investigation of the biological function of individualgenes or the corresponding proteins. The complex network of a cellcannot be characterised simply by decoding the genomic DNA of a humanbeing. The genomic analysis must be followed by an investigation of theproteins encoded by the genome, since it is only with this additionalinformation that the dynamic functioning of the human organism can bedescribed at a molecular level. In addition, there is frequently only aminor correlation between gene transcription and the correspondingtranslation product, and consequently it is only with the aid ofproteome analysis that it can be determined which proteins are expressedto what extent and possibly modified post-translationally under giveninfluences (Allen, L.: Functional Genomics Nature 405 (2000) 819-865;Ezzell, C.: The Business of the Human Genome Scientific American July(2000) 39-57).

In order to be able to investigate proteins, for example, with respectto their expression pattern or their function, they first have to bemade available for the corresponding analyses.

Parallel separation and analysis of all proteins in a cell is virtuallyimpossible using today's methods. A human cell expresses between 35,000and 50,000 proteins, which vary in abundance by a factor of 10⁶. Eventhe protein separation technique that currently has the highestresolution, two-dimensional gel electrophoresis, can separate a maximumof 5,000 to 10,000 proteins with high resolution, with it being possibleprincipally to visualise the proteins having a high expression rate froma complete lysate. However, since in most cases low-abundance proteinstend to be more involved in pathological processes, fractionation of theproteins in a cell into sub-units, i.e. the production of partialproteomes, is necessary from clinical and diagnostic points of viewbefore the protein analysis.

In order to clarify the function of a protein, various basic techniques,such as, for example, activity assays or protein interaction analyses,have been developed. However, these usually require that the proteins tobe analysed are provided in native, non-denatured state for therespective functional analysis.

A valuable supplementary approach for investigating the function ofproteins is analysis of the subcellular localisation of proteins or theredistribution thereof in a cell. Many regulation processes in a cellresult in modification of the subcellular localisation after interactionwith a signal molecule, with a change in RNA transcription being caused,for example by transfer of a protein from the cytosol into the cellnucleus.

There is thus increasing demand for standardised methods for theproduction of partial proteomes which on the one hand cause a reductionin the total number of proteins to be analysed in parallel and on theother hand allow the subcellular localisation (topology) to be includedas additional information in the functional proteome analysis.

Various methods are known for the production of partial proteomes, but,due to the type of purification, they each only make part of theproteome available for analysis. Examples of such methods are thepurification of separated cell compartments based either on differentnet charge or different density of the compartments, on differentprotein composition, which can be utilised for affinity purification, oron different solubility of the organelle proteins in the presence ofspecific buffers.

Since only part of the complete proteome of a cell is made available foranalysis in these methods, proteins which are not present in theparticular purified partial proteome are not available for furtheranalysis. In addition, the investigation of a topological redistributionof proteins is not possible. Prior separation of the sample collectiveand parallel production of different partial proteomes can only overcomethe above-mentioned disadvantages to a limited extent, since thisvariant is firstly very labour-intensive and secondly entails the riskthat the samples are not homogeneous. In addition, the amount of sampleavailable is frequently so small that splitting of the sample isimpossible.

It would therefore be desirable to have a method which enables theentire sample to be separated into various partial proteomes in asequential method, with all proteins of the sample ultimately beingavailable for further analyses as a constituent of one of the partialproteomes.

A known method for the sequential purification of a plurality of cellcompartments is selective detergent extraction [1, 2], in which fourfractions (partial proteomes) are obtained. In the first step, acytosolic fraction is obtained, in which the soluble proteins of thecytosol and soluble components of the cytoskeleton are enriched. In thesecond step, a fraction containing proteins of the membrane/organellesis obtained. The third step gives a ‘nuclear’ fraction, in which,according to the authors, proteins of the nuclear membrane and solublenuclear proteins are enriched. In the fourth and final step, a fractioncontaining proteins of the detergent-resistant cytoskeleton and of thenuclear matrix is formed.

Since the date of publication of the original method in 1994, variousmodifications have been introduced, all of which have concentratedexclusively on the final extraction step after solubilisation of thecell nucleus (summarised in Patton, 1999 [3]). The method described wasemployed for the investigation of the subcellular redistribution of atranscription factor [4] which is involved in signal transduction bycytokines and, after cytokine-induced translocation into the nucleus, isable to bind directly to the DNA [5, 6, 7]. According to the authors'results, however, soluble and/or DNA-associated cell nuclear proteins,in particular, together with proteins of the cytoskeleton and nuclearmatrix were only obtained in the final and denaturing extraction step.

This means that the methods known to date for the sequentialpurification of a plurality of cell compartments by selective detergentextraction [1, 2, 3] do not enable unambiguous topological assignment ofnuclear proteins since an independent fraction separated from componentsof the cytoskeleton and containing, in particular, the proteins from thecell nucleus interior is not obtained.

A further disadvantage of the known extraction method is that thetranscription factors are also present in the fourth fraction. Sincetranscription factors typically occur in a significantly smaller amountthan cytoskeleton proteins, their detection and analysis is madesignificantly more difficult by the presence of the cytoskeletonproteins.

In addition, subsequent functional analysis of the proteins present inthe fourth fraction is greatly restricted since sodium dodecylsulfate(SDS) is employed in the isolation of this fraction. This denatures theproteins, meaning that they are no longer available for importantanalytical methods, such as, for example, co-immunoprecipitation, enzymeactivity tests or electrophoretic mobility shift assay (EMSA). This islikewise a crucial barrier to subsequent functional analysis of thisimportant regulatory protein class.

These problems were not recognised in any of the applications carriedout since 1994 or modifications to the extraction protocol, let aloneimprovements proposed in this respect.

In eukaryotic cells, however, it is precisely the proteins of the cellnucleus that are of major importance for a very wide variety offunctions of the cell. The cell nucleus makes up about 10% of the totalvolume of a eukaryotic cell and is surrounded by a nuclear sheathconsisting of two membranes. The nuclear sheath is connected directly tothe endoplasmatic reticulum and is connected to two networks ofintermediary filaments: firstly a thin layer of filaments (nuclearlamina) in the cell nucleus interior, which supports the inner cellmembrane, and secondly less regularly organised intermediary filamentswhich surround the outer nuclear membrane. These intermediary filamentnetworks thus give the nuclear sheath a certain stability. The nuclearsheath surrounds the nucleoplasm, which contains, inter alia, chromatin(DNA and proteins associated therewith) and the nucleolus. The nuclearproteins in the nucleoplasm, i.e. in the cell nucleus interior, include,in particular, DNA-binding proteins, such as histones and non-histoneproteins (for example HMGs and transcription factors), RNA-bindingproteins, which are involved in RNA splicing and transport, andskeleton-associated proteins. The proteins which form the skeleton inthe cell nucleus interior, in particular the lamina, are, in thisdescription, not regarded as part of the nucleoplasm or of the cellnucleus interior. In particular, DNA-binding proteins, such as histonesand non-histone proteins (for example HMGs and transcription factors),RNA-binding proteins, which are involved in RNA splicing and transport,and skeleton-associated proteins are therefore regarded below asproteins from the cell nucleus interior.

Various methods are described in the literature for the separatesolubilisation of cell nucleus proteins, inter alia using high saltconcentrations (≧1 M), it being known that high salt concentrations inthe extraction buffer interfere with protein interactions and thus withthe further functional analyses, such as, for example, analysis ofprotein complexes. In addition, high salt concentrations in theextracted fractions impair all subsequent protein analysis methods, suchas, for example, isoelectric focusing (IEF), enzyme activity tests,EMSA, ELISA, SDS-PAGE and 2DE. Furthermore, high salt concentrationsreadily attack cytoskeleton and cytoskeleton-associated proteins, whichcan cause contamination of the cell nucleus protein fraction bycytoskeleton proteins.

The object of the present invention was therefore to provide a methodwhich, in the course of sequential separation of the complete proteomeinto partial proteomes, also facilitates the production, in addition tofurther partial proteomes, of a partial proteome of the proteins fromthe cell nucleus interior without significantly denaturing the proteinsfrom the cell nucleus interior in the process.

It has been found that, during the production of partial proteomes fromthe complete proteome of a cell preparation, from the cell nucleuspreparation obtained, inter alia, after removal of the cytosolicproteins and the proteins of the membrane/organelles, the liberation ofproteins from the cell nucleus interior is effected efficiently andsurprisingly simply without significantly impairing the integrity of thecytoskeleton if the cell nucleus preparation is extracted with a saltand detergent solution according to the invention and preferablyadditionally with a nuclease.

Since the proteins of the detergent-resistant cytoskeleton and thenuclear matrix are not co-extracted to a significant extent duringpreparation of the partial proteome of the proteins from the cellnucleus interior, these can be obtained as an independent fraction in asubsequent sequential method step.

The present invention therefore relates to a method for the sequentialproduction of partial proteomes from the complete proteome of a cellpreparation, characterised by the following method steps:

-   -   a) provision of a sample containing a cell preparation    -   b) extraction of the cytosolic proteins and the        membrane/organelle proteins from the sample provided in step a),        leaving a cell nucleus preparation    -   c) extraction of the proteins from the cell nucleus interior        from the cell nucleus preparation obtained in step b) by        treatment with an extraction buffer having a pH of between 6.5        and 8 which comprises at least the following constituents:        -   in total from 0.1 to 7 per cent by weight of one or more            nonionic detergents        -   in total from 0.05 to 3 per cent by weight of one or more            cholic acid derivatives        -   one or more salts from the group consisting of the alkali            metal and/or ammonium salts in a total concentration of            between 75 and 500 mmol/l,        -   where detergent-resistant proteins of the cytoskeleton and            of the nuclear matrix are not extracted to a significant            extent together with the proteins from the cell nucleus            interior, but instead remain in the extraction residue. The            extract obtained in step c) is accordingly a partial            proteome enriched with proteins from the cell nucleus            interior.

In a preferred embodiment, the extraction buffer employed in step c)additionally comprises a nuclease.

In a preferred embodiment, the extraction buffer employed in step c)comprises polyoxyethylene sorbitan monopalmitate (Tween® 40) as nonionicdetergent, deoxycholate as cholic acid derivative and NaCl as alkalimetal salt.

In a preferred embodiment, the extraction of the cytosolic proteins andthe membrane/organelle proteins in step b) is carried out by:

-   -   b i) extraction of the cytosolic proteins from the sample        provided in step a) by selective permeabilisation of the plasma        membrane without significantly impairing the integrity of the        subcellular membrane/organelle structures, the cell nucleus and        the cytoskeleton. The extract obtained is a partial proteome        enriched with cytosolic proteins.    -   b ii) extraction of the membrane/organelle proteins from the        part of the sample remaining after the extraction in step b i)        with retention of the structural integrity of cell nucleus and        cytoskeleton. The extract obtained is a partial proteome        enriched with membrane/organelle proteins.

In a preferred embodiment, the proteins of the detergent-resistantcytoskeleton and of the nuclear matrix are, in an additional method stepd), extracted as a further partial proteome from the extraction residueremaining in step c).

The present invention also relates to the use of the method according tothe invention in the investigation of the redistribution of proteins orthe functional analysis of proteins.

The present invention additionally relates to a protein extraction kitat least containing an extraction buffer having a pH of between 6.5 and8 which comprises at least the following constituents:

-   -   in total from 0.1 to 7 per cent by weight of one or more        nonionic detergents    -   in total from 0.05 to 3 per cent by weight of one or more cholic        acid derivatives    -   one or more salts from the group consisting of the ammonium        and/or alkali metal salts in a total concentration of between 75        and 500 mmol/l.

In a preferred embodiment, the kit additionally contains a nucleasewhich can be added to the extraction buffer.

In a preferred embodiment, the kit additionally contains buffer forextraction of the cytosolic proteins and/or the membrane/organelleproteins from cell preparations and a buffer for extraction of theproteins of the detergent-resistant cytoskeleton and of the nuclearmatrix. The method according to the invention can be carried out withthis preferred embodiment of the kit for the production of four partialproteomes from the complete proteome of a cell preparation.

The explanations for FIGS. 1 to 11 are given in Examples 1 to 12.

The method according to the invention facilitates for the first time theseparation of the complete proteome of a cell preparation into aplurality of subcellular partial proteomes, with one of the partialproteomes being enriched with proteins from the cell nucleus interior. Atopological assignment and functional analysis is thus facilitated forthe first time by an extraction method.

For the purposes of the invention, the term partial proteome of theproteins from the cell nucleus interior means a partial proteome inwhich principally proteins from the cell nucleus interior are enriched.The partial proteome of the proteins from the cell nucleus interioraccording to the invention comprises no or only a small proportion,which does not interfere with the further functional analyses, ofdetergent-resistant proteins of the cytoskeleton and of the nuclearmatrix. Furthermore, the proteins in the partial proteome of theproteins from the cell nucleus interior obtainable by the methodaccording to the invention are in substantially non-denatured form. Thismeans that denaturing of the proteins by the extraction buffer employedin step c) in accordance with the invention is avoided so substantiallythat an adequate proportion of the partial proteome obtained in step c)can be provided for further functional analysis.

The cell preparation employed in the method according to the inventioncan be eukaryotic cells, for example cells cultivated in suspension oradherent cells. Cell preparations from tissue can also be employed,giving cells or cell groups, by dividing tissue by means of biochemical,enzymatic and/or physical means. The cell preparation may also compriseother eukaryotic cells, such as, for example, spheroplasts from yeastcells, plant cells after removal of the cell wall or insect cells, andcell preparations from fungi. The cell preparation employed in themethod according to the invention preferably comprises eukaryotic tissueculture cells from mammals.

The term cell nucleus preparation is applied in accordance with theinvention to residues of the cell preparation from which the cytosolicproteins and the membrane/organelle proteins have been removed inadvance. During removal of these proteins, it must be ensured that thecell nuclei present in the resultant cell nucleus preparation are stillintact to the extent that the proteins from the cell nucleus interiorare still predominantly in the cell nucleus preparation and have notalready been removed during the removal of the cytosolic proteins andthe membrane/organelle proteins. This condition is ensured, for example,by the buffer systems recommended below for the extraction of thecytosolic proteins and the membrane/organelle proteins. Furthermore, acell nucleus preparation prepared by removal of the cytosolic proteinsand the membrane/organelle proteins from cell preparations usually alsocomprises constituents of the cytoskeleton.

The method according to the invention facilitates the division of thecomplete proteome of the cell preparation into at least three partialproteomes, one of which is enriched with the proteins from the cellnucleus interior. Besides the partial proteome of the proteins from thecell nucleus interior, various other partial proteomes may be produced.The nature of the further partial proteomes is dependent on the type ofextraction carried out in step b) and the extraction steps carried outafter the extraction of the partial proteome of the proteins from thecell nucleus interior (step c).

For example, the cytosolic proteins and membrane/organelle proteinsextracted in step b) can be extracted in parallel in the form of apartial proteome or preferably sequentially, giving two partialproteomes (one partial proteome enriched with cytosolic proteins, onepartial proteome enriched with membrane/organelle proteins). For thepurposes of the invention, “enriched” means that the proteins referredto as “enriched” are predominantly present in the particular partialproteome. Other proteins are only present in an amount which does notsignificantly impair further analysis of the enriched proteins.

In a preferred embodiment, the selective permeabilisation of the plasmamembrane of the cells in step b) for extraction of the cytosolicproteins is effected without significant impairment of other cellularsub-structures by chemical treatment with a saponin which ispreferentially incorporated into cholesterol-rich membranes, inparticular with a digitonin-containing buffer. Alternatively, this couldalso be effected by means of other substances, such as, for example,streptolysin-O, enzymatic treatment with, for example, lipase or theaction of mechanical forces by means of, for example, electroporation,freezing/thawing, filter ripping, or combinations thereof. Such methodsare described in the literature and are known to the person skilled inthe art. The extract obtained is a partial proteome enriched withcytosolic proteins.

After extraction of the cytosolic proteins, the membrane/organelleproteins are extracted from the extraction residue with substantialretention of the structural integrity of cell nucleus and cytoskeleton.Suitable for this purpose are, for example, nonionic detergents orzwitterionic detergents under mild conditions. In particular, Triton®X-1 00 is suitable for this purpose. These methods are also described inthe literature and are known to the person skilled in the art. Theextract obtained is a partial proteome enriched with membrane/organelleproteins. The extraction residue obtained is a cell nucleus preparation,which can be passed on to method step c).

If the properties of the cell nucleus preparation with respect toretention of the structural integrity of the cell nucleus or cell nucleifor step c) are not significantly changed, the extraction of thecytosolic and membrane/organelle proteins in step b) can also be carriedout with other agents. It is furthermore possible also to carry out stepb) in more than two extraction steps in order to produce more than twopartial proteomes. However, separation in two sub-steps to produce apartial proteome which is enriched with cytosolic proteins and a partialproteome which is enriched with membrane/organelle proteins ispreferred.

In order to produce only one partial proteome which comprises both thecytosolic and the membrane/organelle proteins in step b), it issufficient to treat the cell preparation from step a) with reagents asdescribed above for the extraction of the membrane/organelle proteins,i.e. with nonionic detergents or zwitterionic detergents under mildconditions. Preference is given here to detergents having an HLB valueof between 12 and 20 for the non-denaturing solubilisation of membraneproteins. In particular, detergents having a high aggregate number, suchas, for example, Triton® X-100 and NP-40, are suitable for this purpose.Prior treatment for selective extraction of cytosolic proteins (such astreatment with a digitonin-containing buffer or with streptolysin-O,enzymatic treatment with, for example, lipase or the action ofmechanical forces by means of, for example, electroporation,freezing/thawing, filter ripping, or combinations thereof) is generallynot necessary.

The cell nucleus preparation obtained after extraction of the cytosolicproteins and the membrane/organelle proteins is then, in accordance withthe invention, subjected to selective detergent extraction of theproteins from the cell nucleus interior in step c).

For the method according to the invention, it is of great importance instep c), the production of a partial proteome which is enriched withproteins from the cell nucleus interior, that the majority of theproteins from the cell nucleus interior are bound to bonding partners,such as nucleic acids or other proteins. The strength of the bond formedby the proteins from the cell nucleus interior to their respectivebonding partners varies greatly and is partially dependent on theenvironment present. Thus, for example, the interaction of histones withDNA molecules is significantly stronger than that of HMGs. In the methodaccording to the invention, the bonding of the proteins from the cellnucleus interior to their bonding partners, in particular nucleic acids,can be employed in a targeted manner for further selection of theproteins from the cell nucleus interior. Depending on the choice ofextraction buffer, weakly bonded, strongly bonded proteins from the cellnucleus interior or all proteins from the cell nucleus interior can beextracted to an increased extent, virtually independently of theirbonding strength to the respective bonding partners, in particularnucleic acids.

The extraction buffer used in step c) typically has a pH of between 6.5and 8. At lower pH values, problems can arise, in particular, with thesolubility of certain constituents, such as the nonionic detergents orthe cholic acid derivatives. The preferred pH range is between pH 6.9and pH 7.8. Suitable buffer substances are therefore those which bufferin the weakly acidic to weakly alkaline range, such as MOPSO, BES, MOPS,phosphate or preferably PIPES. The buffer concentration is typically2-100 mM, preferably between 5 and 20 mM.

Furthermore, the extraction buffer used in accordance with the inventioncomprises one or more suitable nonionic detergents in a proportion oftypically in total from 0.1 to 7% by weight, in any case above the CMC,preferably between 0.2 and 5% by weight. An increase in the detergentconcentration beyond the stated range entails the risk that increaseddenaturing of proteins occurs. In addition, high detergentconcentrations can interfere with the later analytical methods.

Important features for the differentiation of detergents are, forexample, the HLB value, CMC and the aggregate number.

Nonionic detergents which are suitable in accordance with the inventionare those which, in the concentration selected in accordance with theinvention in combination with the other constituents of the extractionbuffer, have the effect that the cell nucleus structure previouslypresent is dissolved. It is important here that the cell nucleusmorphology previously present is destroyed and the nucleoplasm proteinsare liberated without a significant proportion of the cytoskeletonproteins being liberated. Nonionic detergents which are particularlysuitable in accordance with the invention are, for example, thosecontaining a hydrophilic polyoxyethylene head group containing no phenylring between alkyl chain and head group, preferably Tween® detergents,in particular polyoxyethylene sorbitan monopalmitate (Tween® 40).

A further essential constituent of the extraction buffer used inaccordance with the invention is one or more cholic acid derivatives.Cholic acid derivatives which are suitable in accordance with theinvention are anionic detergents. They have a steroid backbone carryingone or more identical or different side chains, such as, for example,—OH, —CH₃, C₂H₅ or, for example, amino acids, and a carboxyl group atthe end of an alkyl chain. Particularly suitable are cholic acidderivatives which—in contrast to nonionic detergents, such asTriton—have a low aggregate number. For the purposes of the invention,cholic acid derivatives are also taken to mean cholic acid itself andsalts of cholic acid. The cholic acid derivative employed is preferablyNa deoxycholate.

In the extraction buffer employed in accordance with the invention instep c), one or more cholic acid derivatives are used in a proportion oftypically in total from 0.05 to 3 per cent by weight, preferably from0.1 to 2.5% by weight. Here too, an increase in the proportion beyondthe stated range can cause denaturing of the proteins and/orinterference with the further analyses.

The extraction buffer furthermore comprises one or more alkali metaland/or ammonium salts in a concentration of between 75 and 500 mmol/l.An increase in the salt concentration beyond the stated concentrationrange can cause denaturing of the proteins and/or interference with thefurther analyses.

Preference is given in accordance with the invention to alkali metalsalts, in particular sodium salts, such as nitrates, sulfates,phosphates and particularly halides, such as bromides or chlorides. NaClis particularly preferred.

An extraction buffer for step c) which is particularly preferred inaccordance with the invention comprises about 10 mM PIPES, about 1% byweight of Tween® 40, about 0.5% by weight of Na deoxycholate and about350 mM NaCl. In addition, the extraction buffer for step c) may comprisefurther constituents, such as, for example, stabilisers orpreservatives.

In a preferred embodiment of the method, the extraction buffer for stepc) additionally comprises a nuclease, preferably an endonuclease fromSerratia marcescens (Benzonase® from Merck KGaA, Darmstadt). For theactivity of the nucleases, it is usually advantageous additionally toadd alkaline earth metal salts to the extraction buffer, typically in aconcentration of between 0.02 and 10 mmol/l. Particularly suitable saltsand the concentration thereof should be selected depending on thenuclease used. On use of Benzonase®, MgCl₂, for example, in aconcentration of between 1 and 10 mM, in particular between 1 and 2 mM,is suitable.

The suitable amount and activity of the nuclease employed depends on thetarget duration of the extraction step. The more nuclease is added, thefaster the extraction can be carried out. However, it must be borne inmind here that the partial proteome obtained in the extraction is alsocontaminated with the nuclease. The more nuclease is employed, the morenuclease is also present in the partial proteome which is enriched withthe proteins from the cell nucleus interior. It must be noted thatnucleases interfere with certain protein analysis methods, such as, forexample, EMSA. If the partial proteome which is enriched with theproteins from the cell nucleus interior is to be subjected to ananalytical method of this type, the addition of nucleases must beavoided.

As can be seen from Examples 4 and 5, the addition of nuclease caninfluence whether weakly bound or strongly bound proteins from the cellnucleus interior are preferentially extracted. If no nuclease is addedto the buffer, the proportion of strongly nucleic acid-bound proteins,such as the histones, is reduced.

The integrity of the cytoskeleton is not significantly impaired bymethod step c) according to the invention for the production of apartial proteome of the proteins from the cell nucleus interior, meaningthat the method according to the invention can give for the first time afraction enriched in the low-abundance nuclear proteins and separatedfrom the high-abundance components of the cytoskeleton.

In addition, following step c), the proteins of the detergent-resistantcytoskeleton and of the nuclear matrix can be extracted from theremaining extraction residue as a further partial proteome by knownmethods in a subsequent extraction step. Suitable for this purpose are,for example, SDS-containing denaturing buffers. These buffers mayadditionally comprise reducing agents, such as, for example, DTT orβ-mercaptoethanol.

The method according to the invention is simple, efficient andautomatable and significantly simplifies the detection and analysis ofthe low-abundance and simultaneously pharmacologically most interestingregulatory protein classes (membrane proteins, such as, for example,receptors on the plasma membrane or DNA-associated proteins andtranscription factors). This is achieved by simple and selective removalof high-abundance proteins (for example household proteins in thecytosol and also cytoskeleton proteins). The method allows for the firsttime the examination of functional proteins of the cell nucleusseparately from the cytoskeleton without the loss of other partialproteomes. The partial proteomes obtained can be subjected to amultiplicity of established protein analysis techniques. This alsoallows, for example, functional studies by means of enzyme activityassays or electrophoretic mobility shift assay (EMSA) for cell nuclearproteins. In addition, the method according to the invention facilitatesnot only the topological assignment of cellular proteins, but also theanalysis of the dynamic redistribution of the said protein classes intothe various cellular compartments, including the cell nucleus, which wasnot possible by previous approaches. An analysis of this type providesimportant information on the function of the protein investigated.Linked to common techniques, such as, for example, mass spectrometry(MS), 2D gel electrophoresis (2DE), amino acid sequencing orimmunoblotting, comprehensive analysis of the proteins present in thecell and the subcellular distribution thereof can be employed in orderto identify novel proteins or protein functions and to indicate theirinvolvement in certain cellular signal pathways. The method according tothe invention thus represents a fundamental approach for characterisingboth physiological and also pathophysiological cellular processes orchanges which arise, inter alia, due to diseases or under the influenceof medicaments/substances.

The present invention additionally relates to a protein extraction kit,in particular for the production of partial proteomes from a completeproteome present in a cell preparation, at least containing anextraction buffer having a pH of between 6.5 and 8, which at leastcomprises the following constituents:

-   -   in total from 0.1 to 7 per cent by weight of one or more        nonionic detergents    -   in total from 0.05 to 3 per cent by weight of one or more cholic        acid derivatives    -   one or more salts from the group consisting of the ammonium        and/or alkali metal salts in a total concentration of between 75        and 500 mmol/l.

The preferred amounts of the individual constituents indicated for theextraction buffer for step c) likewise apply to the extraction buffer inthe kit. The buffer is typically in the form of a dry reagent mixturefor dissolution in water, in the form of an aqueous concentrate or fordirect use in aqueous form.

In a preferred embodiment, the kit furthermore contains a nuclease foraddition to the extraction buffer.

In a preferred embodiment, the kit additionally contains buffers forextraction of the cytosolic proteins and/or the membrane/organelleproteins from cell preparations. The kit particularly preferablycontains reagents for carrying out the preferred embodiment of themethod according to the invention in which four partial proteomes(partial proteome enriched with cytosolic proteins, partial proteomeenriched with membrane/organelle proteins, partial proteome enrichedwith proteins from the cell nucleus interior, partial proteome enrichedwith proteins of the cytoskeleton and of the nuclear matrix) areobtained from the complete proteome of a cell preparation.

The kit may equally have further constituents or reagents, such asequipment and consumables suitable for carrying out the method accordingto the invention and/or reagents for further processing of the partialproteomes, for example for functional analysis or storage.

Even without further comments, it is assumed that a person skilled inthe art will be able to utilise the above description in its full scope.The preferred embodiments and examples should therefore merely beregarded as descriptive disclosure which is absolutely not limiting inany way.

The complete disclosure content of all applications, patents andpublications mentioned above and below, and of the correspondingapplication EP 02019570.7, filed on 02 Sep. 2002, is incorporated intothis application by way of reference.

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[12] Dignam et al. (1983):“Accurate transcription initiation by RNApolymerase II in a soluble extract from isolated mammalian nuclei”Nucleic-Acids-Research 11(5), 1475-89 List of abbreviations μ micro 2-DEtwo-dimensional gel electrophoresis A431 epithelial carcinoma cells BesN,N-bis(2-hydroxyethyl)-2-aminoethane- sulfonic acid CHO Chinese hamsterovary CMC critical micelle concentration DAPI4′,6-diamidino-2-phenylindole DNA desoxyribonucleic acid DOCdeoxycholate DTT dithiothreitol EDTA ethylenediaminetetraacetic acidELISA enzyme linked immunosorbent assay EMSA electrophoretic mobilityshift assay F1 fraction 1, enriched cytosolic proteins F2 fraction 2,enriched membrane/organelle proteins F3 fraction 3, enriched nuclearproteins F4 fraction 4, enriched cytoskeleton proteins H1, H2, . . . ,H4 histone 1, histone 2, . . . , histone 4 HEPES[4-(2-hydroxyethyl)piperazino]ethane- sulfonic acid HLB valuehydrophilic/hydrophobic balance HMG high motility group HSP70 heat-shockproteins 70 kDa IEF isoelectric focusing KDa, kD kilodaltons L litres Mmolar mA milliamperes MCF7 mammacarcinoma cells Mes2-morpholinoethanesulfonic acid mg milligrams MgCl₂ magnesium chloridemin minutes mio million mmol/l millimoles per litre MOPS3-morpholino-1-propanesulfonic acid MOPSO3-(N-morpholino)-2-hydroxypropane- sulfonic acid MS mass spectrometry mVmillivolts MW molecular weight NaCl sodium chloride Na-DOC sodiumdeoxycholate NP-40 polyethylene glycol p-isooctyl phenyl ether plisoelectric point PIPES piperazine-1,4-bis(2-ethanesulfonic acid) PVDFpolyvinylidene difluoride RNA ribonucleic acid RT room temperature SAOS2 osteosarcoma cells SDS sodium dodecylsulfate SDS-PAGE sodiumdodecylsulfate polyacrylamide gel electrophoresis T₂₅ culture culturebottle with an area of 25 cm² bottle T₇₅ culture culture bottle with anarea of 75 cm² bottle tris 2-amino-2-(hydroxymethyl)-1,3-propane- diolTriton ® X-100 octylphenoxypolyethoxyethanol Tween ® 40 polyoxyethylenesorbitan monopalmitate U unit ON overnight V volts v/v volume/volume w/vweight/volume w/w weight/weight WB western blotting

EXAMPLES Example 1 Investigation of the Protein Pattern by Means ofSDS-PAGE of Extracts After Selective Detergent Extraction of EukaryoticCells in Accordance with the Prior Art [1, 2, 3]

The starting material for this experiment was mammacarcinoma cells(MCF7) which were confluent to the extent of 80% as adherent cell lawnon a tissue culture dish. The extraction steps were carried out inaccordance with the prior art. In this way, protein extracts F1-F4 wereobtained. These protein extracts were separated volume-equivalently bymeans of a 12% polyacrylamide gel in an electric field and visualisedusing Coomassie Brilliant Blue. An example of this type is shown by FIG.1.

The following terms are used in FIG. 1:

M=marker. F1 denotes fraction 1 containing the cytosolic proteins. F2stands for fraction 2 and represents the membrane/organelle proteins. F3demonstrates the fraction 3 referred to in the prior art [1, 2, 3] as‘nuclear’ fraction, and F4 signifies fraction 4, which, in accordancewith the prior art, is said to contain the proteins of the cytoskeletonand of the nuclear matrix.

A striking feature of the results obtained on reproducing the prior art(see FIG. 1) is the very low protein content of the ‘nuclear’ fraction 3compared with fractions 1, 2 and 4. Furthermore, the typical bandpatterns of the DNA-associated histone proteins (which belong to thegroup of proteins from the cell nucleus interior) are present infraction 4, together with the proteins of the cytoskeleton. The typicalband patterns of the DNA-associated histone proteins are denoted by an *in FIG. 1.

This result supports the ascertainment of the fact that the methoddescribed in the literature [1, 2, 3] for the production of four partialproteomes does not give a partial proteome in which the proteins fromthe cell nucleus interior are enriched clearly separated from theproteins of the cytoskeleton. The fraction 3 obtainable in accordancewith the prior art is not enriched with the proteins from the cellnucleus interior. Rather, the majority of these are located in fraction4, in which on the one hand the proteins are only obtained in denaturedform, and on the other hand the proteins of the cytoskeleton areextracted.

Example 2 Immunoblotting Investigation of the Topological Assignment ofMarker Proteins in Extracts After Selective Detergent Extraction ofEukaryotic Cells in Accordance with the Prior Art [1, 2, 3]

The protein extracts obtained in accordance with Example 1 wereseparated by means of SDS-PAGE and subsequently blotted on a PVDFmembrane and investigated using selected marker proteins.

The marker proteins of the cytosol (a-b) and of the membrane/organelles(b-d) are detected in the corresponding fractions (F1 to F4) afterextraction of a cell preparation in accordance with the prior art (cf.FIG. 2). However, marker proteins from the cell nucleus interior are notobtained in the corresponding fraction F3, referred to as ‘nuclear’fraction, after extraction of a cell preparation in accordance with theprior art. The transcription factor c-jun (e) and the DNA-associatedprotein histone 1 (f) (both proteins from the cell nucleus interior) arenot detected in the ‘nuclear’ fraction F3, but instead are onlyliberated in the final fraction (F4) together with the high-abundancecytoskeleton marker cytokeratin (g) under denaturing conditions.

a: calpain; b: HSP 70; c: cadherin; d: cytochrome P 450 reductase; e:c-jun; f: histones; g: cytokeratin

The immunoblot analysis of the fractions obtained for representativemarker proteins after detergent extraction in accordance with the priorart demonstrates that an independent fraction is not obtained for thesoluble and DNA-associated proteins from the cell nucleus interior.Rather, these are solubilised under denaturing conditions together withproteins of the cytoskeleton. Thus, for example, clear analysis of thetopological redistribution of the proteins from the cell nucleusinterior is not possible using the method in accordance with the priorart.

Example 3 Illustrative Description of Selective Detergent Extraction bythe Method According to the Invention

The extraction buffers were prepared in accordance with the table under3a. The extraction is carried out as described in detail under 3b. Theresults of the method according to the invention are described ingreater detail in the following examples.

a. Buffer Composition Used for the Experiment Documented in 3b. BufferChemicals Concentration EXTRACTION PIPES 10 mM BUFFER I Digitonin 0.02%pH 6.8 Sucrose 300 mM Sodium chloride 15 mM EDTA 0.5 mM EXTRACTION PIPES10 mM BUFFER II Triton X-100 0.50% pH 7.4 Sucrose 300 mM Sodium chloride15 mM EDTA 0.5 mM EXTRACTION PIPES 10 mM BUFFER III Tween-40  1.0% pH7.4 Deoxycholate  0.5% Sodium chloride 350 mM Magnesium chloride 1 mMBenzonase 500 U/ml EXTRACTION Sodium dodecylsulfate (SDS)   5% BUFFER IVNa2HPO4 10 mM pH 7.4 NaH2PO4 10 mM

b. Illustrative Performance of the Extraction of Adherent Cells

The subcellular fractionation was carried out using tissue culture cellsin T₂₅ tissue culture dishes. If other culture vessels are used, theamounts of buffer can be scaled up or down correspondingly to the areaof the plate used. For the extraction, vital cells in the logarithmicgrowth phase at about 80% confluence must be employed. In this example,SAOS 2 cells were used.

-   -   1. Thaw extraction buffers I, II and III on ice, extraction        buffer IV and the protease inhibitor cocktail at RT.    -   2. Slightly tilt the tissue culture dish so that the medium can        be removed from the culture bottle without touching the cell        lawn. Carefully remove media residues.    -   3. Slightly tilt the tissue culture dish. Carefully pipette in 2        ml of ice-cold washing buffer from the edge, covering the cell        lawn. Incubate the culture dish at 4° C. for 5 min while shaking        gently.    -   4. Slightly tilt the tissue culture dish and carefully remove        the washing buffer without touching the cell lawn.    -   5. Repeat steps 3 and 4 in order completely to remove medium        constituents, such as, for example, BSA, amino acids and        indicator dyes.    -   6. Mix 1 ml of ice-cold extraction buffer I and 5 μl of protease        inhibitor cocktail with one another. Carefully pipette onto the        washed tissue culture dish from the edge without touching the        cell lawn. Carefully cover the cells with the buffer. Incubate        at +4° C. for 10 min while shaking gently.    -   7. Carefully tilt the tissue culture dish. Carefully remove the        cytosolic protein extracts (fraction 1) from the culture bottle        without touching the cell lawn and store on ice until used on        the same day. For long-term storage, aliquot in suitable amounts        (for example 100 μl) and store at −80° C.    -   8. Mix 1 ml of ice-cold extraction buffer II and 5 μl of        protease inhibitor cocktail with one another. Carefully pipette        into the tissue culture dish from the edge without touching the        cell lawn. Carefully cover the cells with the buffer, then        incubate at +4° C. for 30 min while shaking gently.    -   9. Carefully tilt the tissue culture dish. Carefully remove the        extract containing the membrane/organelle proteins (fraction 2)        from the culture bottle without touching the cell lawn and        incubate on ice until used on the same day. For longer-term        storage, aliquot in suitable amounts and store at −80° C.    -   10. Mix 500 μl of ice-cold extraction buffer III, 5 μl of        protease inhibitor cocktail and 10 μl (250 U) of Benzonase® with        one another and carefully pipette onto the remaining cell        constituents. Incubate at 4° C. for 10 min while shaking gently.    -   11. Carefully tilt the tissue culture bottle. Carefully remove        the protein extract containing the nuclear proteins (fraction 3)        from the culture bottle without touching the cell lawn and store        on ice until used on the same day. For long-term storage,        aliquot in suitable amounts and store at −80° C.    -   12. Mix 500 μl of extraction buffer IV adjusted to room        temperature and 5 μl of protease inhibitor cocktail with one        another and add to the remaining cell lawn. The cell layer        detaches from the plate on addition of this buffer.    -   13. Suspend by pipetting up and down using a 1 ml pipette tip        (for example Eppendorf). After complete dissolution of the        remaining cell constituents, transfer into a microcentrifuge        tube and store on ice until used on the same day. Fraction 4        contains cytoskeleton proteins. For long-term storage, aliquot        in suitable amounts and store at −80° C.

Example 4 Morphological Portrayal of the Cells When Carrying Out theSelective Detergent Extraction in Accordance with the Invention

The protein extraction was carried out analogously to Example 3 from acell culture in a T₇₅ culture bottle (grown 80% confluently with SAOS 2cells). These were investigated microscopically for morphologicalchanges as a consequence of the method before the extraction, i.e. inuntreated form (picture i), and stepwise after the respective selectivedetergent extraction (pictures ii-iv).

FIG. 3 shows an example of this type. After treatment of the cells withextraction buffer I (picture ii), the cytosolic contents of the cellsescape, whereupon the cells shrink. The integrity of the cell nuclei andplasma membrane is substantially retained. After incubation of the cellswith extraction buffer II (picture iii), the membrane/organelle proteinsare extracted without impairing the integrity of the cell nuclei. Afterincubation of the remaining cell material with extraction buffer III(method step c) according to the invention) (picture iv), the fractionof the proteins from the cell nucleus interior is extracted. Theremaining cell material is taken up in extraction buffer IV, giving afraction containing the cytoskeleton constituents.

P I: extraction buffer I

P II: extraction buffer II

P III: extraction buffer III

P IV: extraction buffer IV

i: SAOS 2 cells, untreated

ii: SAOS 2 cells treated with extraction buffer I, incubation givesfraction 1 (F1)

iii: SAOS 2 cells treated with extraction buffer II, incubation givesfraction 2 (F2)

iv: SAOS 2 cells treated with extraction buffer III, incubation givesfraction 3 (F3)

Example 5 Protein Distribution in Extracts After Selective DetergentExtraction of Eukaryotic Cells in Accordance with the Prior Art [1, 2,3] Compared With the Method According to the Invention

In the method according to the invention, the concentration of an alkalimetal salt in extraction buffer III was increased in this examplecompared with the prior art [1, 2, 3], and in addition a nuclease wasemployed. In order to document the improvement achieved, extractionbuffer III was prepared with varying salt concentrations (prior art, 15mM NaCl +Benzonase®, 150 mM NaCl+Benzonase® and 350 mM NaCl+Benzonase®).The Benzonase® (nonspecific endonuclease) was employed in the sameconcentration in all experiments. All other parameters correspond tothose indicated in the prior art. The biological material employed was 4times about 80% confluent SAOS 2 cells. The protein extracts obtainedwere analysed and evaluated by means of a protein determination methodbased on the Lowry assay.

The method according to the invention indicates more efficientextraction of the cell nucleus. This is clearly evident from asignificant increase in the protein content of the nuclear fraction (F3)and a decrease in the amount of protein in the cytoskeleton fraction(F4).

FIG. 4 shows the results of the protein determination and thedistribution thereof over the four fractions.

The increase in the salt concentration in buffer III enables aredistribution of proteins from fraction 4 (F4) into fraction 3 (F3) tobe achieved in connection with the action of Benzonase®. As expected, nosignificant change in fractions F1 and F2 is observed.

a) Prior art

b) 15 mM NaCl in buffer III +Benzonase (prior art+Benzonase)

c) 150 mM NaCl in buffer III +Benzonase

d) 350 mM NaCl in buffer III +Benzonase

Example 6 Investigations of the Action of the Extraction BufferAccording to the Invention (buffer III) with and without Addition of aNuclease

The distribution of marker proteins for the corresponding fractions waslikewise investigated in the protein extracts from Example 5 by means ofimmunological methods after separation by SDS-PAGE and blotting on aPVDF membrane.

The analysis shown in FIG. 5 demonstrates the distribution oftranscription factors (through the example of c-jun FIG. 5A) andDNA-associated proteins (through the example of histone 1 FIG. 5B) infractions after selective detergent extraction depending on the NaClconcentration and the action of a nuclease. The integrity of thecytoskeleton is not significantly impaired.

In order to test the action of the Benzonase®, the behaviour of thetranscription factors and histone proteins was investigated in thepresence of 350 mM NaCl in extraction buffer III, with and without theaction of nuclease in fraction 3 (FIG. 5C). The use of nuclease withconstant NaCl concentration facilitates a clearly increased recoveryrate of the DNA-associated histone proteins in fraction 3, whereas thetranscription factors can be extracted in the presence of 350 mM NaClwithout the action of nuclease.

Detailed description of FIG. 5:

5A) The recovery rate of the transcription factor c-jun in fraction 3 isdependent on the NaCl concentration in buffer III. From a concentrationof 150 mM NaCl in buffer III, c-jun can be detected in fraction 3 (I:c-jun/15 mM NaCl; II: c-jun/150 mM NaCl; III c-jun/350 mM NaCl). None ofthese conditions impairs the integrity of the cytoskeleton, which isdocumented by the exclusive detection of cytokeratin in fraction 4 (IV:cytokeratin/350 mM NaCl). In all experiments, Benzonase® was employed.On extraction in accordance with the prior-art protocol [1, 2, 3], c-junis detected in the fourth fraction (I).

5B) The behaviour of histone 1 at different salt concentrations inbuffer III. The DNA-associated protein histone 1 was only detected at350 mM NaCl in buffer 3 and simultaneous nuclease action in fraction 3(I: histone 1/15 mM NaCl; II: histone 1/150 mM NaCl; III: histone 1/350mM NaCl). On extraction in accordance with the prior-art protocol [1, 2,3], histone 1 is found in the fourth fraction (I).

5C) c-jun is detected in the presence of 350 mM NaCl in extractionbuffer Ill, independently of the nuclease action in fraction 3 (I:c-jun/buffer III with Benzonase; II: c-jun/buffer III withoutBenzonase). Histone 1, by contrast, required the simultaneous action ofnuclease in order to be detected together with c-jun in fraction 3 (III:histone/buffer III with Benzonase; IV: histone/buffer III withoutBenzonase). The use of the nuclease at a constant NaCl concentrationfacilitates a clearly increased recovery rate of DNA-associated proteinsin fraction 3.

Example 7 Morphological Portrayal of Cell Compartment/Organelles onPerformance of the Selective Detergent Extraction in Accordance with theInvention

The selectivity of the extraction method according to the invention forthe corresponding subcellular partial proteomes was documented byfluorescence microscopy. The protein extraction according to theinvention was carried out with a cell preparation of transfected COS-1cells, otherwise analogously to Example 3. The cells were investigatedfor morphological changes as a consequence of the method by fluorescencemicroscopy before the extraction, i.e. after washing (picture I), andstepwise after the respective extraction step (pictures II-V).

FIG. 6 shows the subcellular compartments or organelles during theextraction according to the invention through the example of COS-1cells. Washing of the cells for the removal of medium constituents whichwould otherwise interfere does not impair the morphology of the cells(picture I). After treatment of the cells with extraction buffer I(picture II), the cytosolic contents of the cells escape, with theintegrity of the cell organelles, of the cell nuclei and of thecytoskeleton being very substantially retained. After incubation of thecells with extraction buffer II (picture III), the membrane/organelleproteins are extracted, with the integrity of the cell nuclei and of thecytoskeleton still being retained. After incubation of the remainingcell material with extraction buffer III (method step c) according tothe invention) (picture IV), the fraction of the proteins from the cellnucleus interior is extracted, with the integrity of the cytoskeletonbeing very substantially retained. The remaining cell material is takenup in extraction buffer IV, giving a fraction containing thecytoskeleton constituents.

F 1: fraction 1

F 2: fraction 2

F 3: fraction 3

F 4: fraction 4

FIG. 6 demonstrates the selectivity of the method according to theinvention with reference to the visualisation of subcellularcompartments or organelles of transfected COS-1 cells by means ofcommercially available fluorescent dyes: phallicidin for staining thefilamentary actin cytoskeleton, DAPI for visualisation of the cellnuclei, ER-Tracker™ for visualisation of the endoplasmatic reticulum,MitoTracker for visualisation of the mitochondria (as membrane/organellemarker) and soluble, green-fluorescent protein (GFP), which is expressedby the transfected COS-1 cells, and is used as marker protein for thecytoplasm.

Description of FIG. 6:

A: phallicidin, B: DAPI staining, C: MitoTracker, D: ER-Tracker™, E: GFP

I: transfected COS-1 cells, untreated

II: transfected COS-1 cells treated with extraction buffer I, incubationgives fraction 1 (F1)

III: transfected COS-1 cells, after treatment with extraction buffer Iadditionally treated with extraction buffer II, incubation givesfraction 2 (F2)

IV: transfected COS-1 cells, after treatment with extraction buffers Iand II additionally treated with extraction buffer III, incubation givesfraction 3 (F3)

V: transfected COS-1 cells, after treatment with extraction buffers I toIII additionally treated with extraction buffer IV, incubation givesfraction 4 (F4)

Example 8 Protein Patterns of Extracts and Topological Assignment ofSubcellular Marker Proteins After Selective Detergent Extraction ofEukaryotic Cells by Extraction Methods According to the Invention

The protein extracts obtained by extraction in accordance with theinvention of SAOS 2 cells as described in Examples 3a and 3b wereseparated by means of SDS-PAGE (10% tricine-PAA gel). The proteins weresubsequently detected using Coomassie Brilliant Blue. A result of thistype is shown in FIG. 7A.

In addition, the protein extracts were blotted on PVDF membranes afterseparation by SDS-PAGE and investigated by immunoblot analysis againstselected marker proteins. The results are shown in FIG. 7B.

In agreement with the protein amount determination in Example 4 and themorphological findings in Example 6, it can be demonstrated here (7A and7B) that efficient liberation of DNA-associated proteins from the cellnucleus interior is facilitated without impairing the integrity of thecytoskeleton in the process. After extraction in accordance with theinvention of a cell preparation as described in Example 3, anindependent fraction of the proteins from the cell nucleus interior isthus obtained, which thus allows for the first time the separateconsideration of functional proteins from the cell nucleus andcytoskeleton without the loss of other partial proteomes.

Detailed description of FIG. 7:

7A) After selective detergent extraction of eukaryotic cells by themethod according to the invention, different protein patterns of theindependent subcellular fractions can clearly be observed. F1 heredenotes fraction 1 containing the cytosolic proteins. F2 stands forfraction 2 and represents the membrane/organelle proteins. F3 (fraction3) corresponds to the proteins from the cell nucleus interior. F4(fraction 4) finally represents the proteins of the cytoskeleton and ofthe nuclear matrix. Compared with extraction in accordance with theprior art [1, 2, 3] (FIG. 2), significantly more proteins are detectedin fraction 3.

7B) Distribution of Marker Proteins Over Subcellular Fractions AfterSelective Detergent Extraction

After selective detergent extraction of eukaryotic cells by the methodaccording to the invention, a clear topological assignment of nuclearmarker proteins is possible, in contrast to the prior art (FIG. 3).Marker proteins of the cytosol and of the membrane/organelles aredetected in the corresponding fractions (a-d). Soluble transcriptionfactors (e and f) and the DNA-associated protein histone 1 (g) areclearly enriched in a fraction of the proteins from the cell nucleusinterior. Only in the final step is the high-abundance cytoskeletonmarker cytokeratin (h and j) extracted separately therefrom.

a: calpain; b: heat-shock protein 70 (HSP 70); c: cadherin; d:cytochrome P 450 reductase; e: c-fos; f: c-jun; g: histones; h:cytokeratin; j: vimentin

Example 9 Protein Patterns of Extracts and Topological Assignment ofSubcellular Marker Proteins After Selective Detergent Extraction ofIntestinal Cancer Tissue by Extraction Methods According to theInvention

Starting from intestinal cancer tissue, cell clusters were produced bystandard methods [8] and treated by the extraction method according tothe invention. Extracts resulting therefrom were separated by means ofSDS-PAGE, transferred to PVDF membranes and investigated by immunoblotanalysis against selected marker proteins. The selected marker proteinsare as follows:

I=calpain, II=cytochrome p 450 reductase, III=c-jun.

In this figure, F1 denotes fraction 1 containing the cytosolic proteins.F2 stands for fraction 2 and represents the membrane/organelle proteins.F3 (fraction 3) corresponds to the proteins from the cell nucleusinterior. The remaining cell material, which can be taken up inextraction buffer IV, was not investigated in this example.

The immunoblot analysis with selected marker proteins confirms thesuitability of selective detergent extraction by extraction methodsaccording to the invention for use with cell preparations from tissueprepared by standard methods [8].

Example 10 Investigation of the Dynamic Subcellular Redistribution ofNFkappaB Which is Involved in Signal Transduction and, After CellularStimulation with TNFalpha from the Cytosol, is Translocated into theCell Nucleus

A431 cells were stimulated with TNFα for various times (0, 5 and 15minutes) and extracted in accordance with the method according to theinvention (see Example 3). The cytosolic fraction (F1), themembrane/organelle fraction (F2), the nuclear fraction (fraction of theproteins from the cell nucleus interior) (F3) and the cytoskeletonfraction (F4) were separated by means of SDS-PAGE for the respectivestimulation time. An immunoblot was subsequently carried out with aspecific antibody against NFkappaB. The time analysis clearlydemonstrates a redistribution of NFkappaB from the cytosol into the cellnucleus. FIG. 9a shows the results of the immunoblot analysis. Theintensity of the protein bands was determined by densitometry. The datameasured were shown graphically in relation to one another (see FIG. 9b).

The method according to the invention is suitable for analysing thedynamic subcellular redistribution of NFkappaB, which is involved insignal transduction and is translocated from the cytosol into the cellnucleus after cellular stimulation with TNFalpha, simply and directly bymeans of immunoblot.

Example 11 The Detection of Endogenous Enzyme Activities After SelectiveDetergent Extraction of Eukaryotic Cells Using the Extraction MethodAccording to the invention Demonstrates Both Selectivity andFunctionality of Representative Enzymes in Extracts I, II and III

After selective detergent extraction of eukaryotic cells by means of themethod according to the invention, enzyme activities of representativemarker enzymes were investigated. The activities for calpain (a), asmarker enzyme for the cytoplasm, for alkaline phosphatase (b), as markerenzyme for membrane/organelles, and for endogenous cellular RNases (c)were determined here. RNases are enzymes which can occur in a pluralityof forms within the cell and may be associated with differentsubcellular compartments depending on the cell type and specificfactors. For protection of cellular RNA, RNase is usually incompartmentalised form. Thus, for example, on the one hand RNase LX [9]and also a number of RNase A subtypes were previously found associatedwith membrane/organelles [10], on the other hand RNase HII is localisedin the cell nucleus [11].

For the determination of the enzyme activity, cell extracts wereemployed which had been prepared in accordance with the invention inaccordance with the procedure in Example 3. In the case of thedetermination of the calpain activity as marker enzyme for thecytoplasm, cell preparations of human skin cancer cells (A 431) wereemployed for this purpose, for the determination of the activity ofalkaline phosphatase as marker enzyme for membrane/organelles (b), cellextracts from SAOS 2 cells were employed. In order to document that thefraction of the cell nuclear proteins is also obtained in the nativestate after extraction in accordance with the invention of cellpreparations, the endogenous activity of the cellular RNases, which alsooccur in the cell nucleus (see above), was determined. In order toenable determination of the activity of these endogenous RNases,additional cell extracts from SAOS 2 cells were prepared, with nonuclease being added to extraction buffer III for this purpose.

The results are shown graphically in FIG. 10, and demonstrate theselectivity of the method according to the invention for cellfractionation in agreement with the morphological findings (FIG. 6) andthe results from the immunoblot analysis (FIG. 7). About 90% of thecalpain activity determined can be detected in the fraction assigned tothe cytoplasm. The activity of the alkaline phosphatase employed asmembrane marker is detected to the extent of more than 70% in themembrane/organelle fraction. The activity of cellular RNases afterextraction in accordance with the invention without the addition ofnuclease is distributed as follows: 54% in the membrane/organellefraction, 30% in the fraction of the cell nuclear proteins and 16% inthe fraction of the cytoskeleton proteins. No RNase activity was foundin the cytoplasm fraction. The extraction buffers employed in accordancewith the invention, particularly those for the production of the partialproteome of the proteins from the cell nucleus interior, yield active,non-denatured proteins. The method according to the invention is thussuitable for the analysis of the subcellular distribution of enzymeactivities.

F1-F4 denote fractions 1-4. The relative enzyme activities for calpain(a), alkaline phosphatase (b) and RNase (c) are shown graphically inthis figure.

Example 12 The Fraction of the Proteins from the Cell Nucleus InteriorContains Functional Transcription Factors Which are Active with Respectto DNA Binding

CHO-K1 cells were extracted by the extraction method according to theinvention (as described in Example 3), but without the addition ofBenzonase® to extraction buffer III (FIG. 11A). A fraction of theproteins from the cell nucleus interior containing active transcriptionfactors, which was obtained by a standard method for the specificpreparation of nuclear extracts [12], was likewise employed as positivecontrol (FIG. 11B).

Samples of the nuclear fractions obtained were subsequently analysed byEMSA in order to determine the binding activity present of transcriptionfactors to an Oct1 oligonucleotide. The analysis clearly demonstratesthat the nuclear fraction contains functional transcription factorswhich are active with respect to DNA binding after the method accordingto the invention. This is an illustrative demonstration of the broadapplicability of the method according to the invention.

Description of FIG. 11:

-   -   1. Fraction of the proteins from the cell nucleus interior        obtained in accordance with the invention    -   2. Fraction of the proteins from the cell nucleus interior        obtained in accordance with the invention+100× non-radioactively        labelled Oct1 sample    -   3. Fraction of the proteins from the cell nucleus interior        obtained in accordance with the invention+100× non-radioactively        labelled SP1 sample    -   4. Fraction of the proteins from the cell nucleus interior        obtained by standard methods    -   5. Fraction of the proteins from the cell nucleus interior        obtained by standard methods+100× non-radioactively labelled        Oct1 sample    -   6. Fraction of the proteins from the cell nucleus interior        obtained by standard methods+100× non-radioactively labelled SP1        sample

1. Method for the sequential production of partial proteomes from thecomplete proteome of a cell preparation, characterised by the followingmethod steps: a) provision of a sample containing a cell preparation b)extraction of the cytosolic proteins and the membrane/organelle proteinsfrom the sample provided in step a), leaving a cell nucleus preparationc) extraction of the proteins from the cell nucleus interior from thecell nucleus preparation obtained in step b) by treatment with anextraction buffer having a pH of between 6.5 and 8 which comprises atleast the following constituents: in total from 0.1 to 7 per cent byweight of one or more nonionic detergents in total from 0.05 to 3 percent by weight of one or more cholic acid derivatives one or more saltsfrom the group consisting of the alkali metal and/or ammonium salts in atotal concentration of between 75 and 500 mmol/l, wheredetergent-resistant proteins of the cytoskeleton and of the nuclearmatrix are not extracted to a significant extent together with theproteins from the cell nucleus interior, but instead remain in theextraction residue.
 2. Method according to claim 1, characterised inthat the extraction buffer employed in step c) additionally comprises anuclease.
 3. Method according to claim 1, characterised in that theextraction buffer employed in step c) comprises polyoxyethylene sorbitanmonopalmitate as nonionic detergent, deoxycholate as cholic acidderivative and NaCl as alkali metal salt.
 4. Method according to claim1, characterised in that the extraction of the cytosolic proteins andthe membrane/organelle proteins in step b) is carried out by: b i)extraction of the cytosolic proteins from the sample provided in step a)by selective permeabilisation of the plasma membrane withoutsignificantly impairing the integrity of the subcellularmembrane/organelle structures, the cell nucleus and the cytoskeleton. bii) extraction of the membrane/organelle proteins from the part of thesample remaining after the extraction in step b i) with retention of thestructural integrity of cell nucleus and cytoskeleton.
 5. Methodaccording to claim 1, characterised in that the proteins of thedetergent-resistant cytoskeleton and of the nuclear matrix are, in anadditional method step d), extracted as a further partial proteome fromthe extraction residue remaining in step c).
 6. Protein extraction kitat least containing an extraction buffer having a pH of between 6.5 and8 which comprises at least the following constituents: in total from 0.1to 7 per cent by weight of one or more nonionic detergents in total from0.05 to 3 per cent by weight of one or more cholic acid derivatives oneor more salts from the group consisting of the ammonium and/or alkalimetal salts in a total concentration of between 75 and 500 mmol/l. 7.Kit according to claim 6, additionally containing a nuclease.
 8. Kitaccording to claim 6 one of claims 6, additionally containing buffer forextraction of the cytosolic proteins and/or the membrane/organelleproteins from cell preparations and a buffer for extraction of theproteins of the detergent-resistant cytoskeleton and of the nuclearmatrix.